Luminescence spectrometry is a pervasive analytical procedure having exceptionally high sensitivity and selectivity. Luminescence measurements are, however, subject to errors caused by quenching species present in the sample under investigation. Such species reduce the luminescence signal and, if not compensated for, result in low concentration estimates for the luminescent species. Problems of quenching are especially severe in complex media such as biological, mineralogical, or environmental samples where the nature and amounts of quenching species are often unknown and not readily controlled.
There are several approaches to the elimination of luminescence quenching effects: dilution, standard addition, and back extrapolation of luminescence decays or correction for the degree of quenching. In the dilution approach the sample is diluted until the quenching species concentration is too low to affect the luminescence intensity. In the standard addition method a known concentration of the luminescent species is added, and the emission intensity is remeasured. Since the luminescence of the standard is quenched to the same extent as that of the unknown, the concentration of the unknown can be readily inferred. In the decay method the sample is excited with a short-duration light pulse, and the decay curve is extrapolated back to the time of the exciting flash. The extrapolated signal may be related directly to the concentration of the luminescent species and is independent of quenching. Alternatively, the observed intensity is corrected for quenching from the measured lifetime.
All three methods have weaknesses and merits. All fail if the quenching is too large. Sample purification before analysis is then mandated. Dilution is simple, but requires additional sample handling and is suitable only if the concentrations of the luminescent species are sufficiently high so that dilution does not reduce the signal intensity below the analytically useful range. Finally, without a priori knowledge of the degree of quenching, the degree of dilution must be established experimentally.
Standard addition does not depend on knowledge of the extent of quenching. However, sample handling is relatively labor intensive since it requires at least two measurements, and the method does not readily lend itself to on-line processing.
The decay methods avoid additional sample handling and eliminate errors arising from sample scatter and from short-lived luminescence. However, the instrumentation is elaborate and expensive, and the significant computational requirements often prevent its use for real-time analytical procedures.
Phase-resolved spectroscopy has been used to reduce interfering signals in the determination of the concentration of one or more components of interest by means of direct supression or resolution of the unwanted signal such as scattered light or fluorescent background. For mixtures of emitters having different fluorescent lifetimes, the total phase-resolved intensity is the sum of the individual contributions. The advantage of phase-resolved fluorimetry is that multiple detector phase angles can be used instead of or in addition to multiple wavelengths to generate sufficient information for the determination of multiple unknown concentrations. Even components with identical spectra can be simultaneously determined by the use of different detector phase angles, provided the fluorescing species have sufficiently different fluorescence lifetimes. The basis of this technique is the use of an excitation beam that is modulated at a high frequency. If a mixture of two emitting species A and B is measured with the detector 90.degree. out-of-phase (in quadrature) with component A, for example, the individual spectrum of component B can be obtained. Essentially all of the work performed with phase-resolved fluorescence spectroscopy has involved the resolution of the individual spectra in multicomponent systems. Recently, however, J. N. Demas and R. A. Keller in "Enhancement of Luminescence and Raman Spectroscopy by Phase-Resolved Background Suppression," Anal. Chem. 57, 538 (1985) have suppressed both fluorescence interference in Raman spectra and scattered light interference in fluorescence spectra for cases in which one signal component is very weak relative to the other. There has been no work to date on the use of modulation methods for reducing the effects of chemical species which quench luminescence.
Accordingly, it is an object of the present invention to substantially reduce the effects of quenching species on the luminescence of species of interest.
Another object of the present invention is to provide a method for the quantitative analysis of species by luminescence spectrometry in the presence of quenching species.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.